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Science Highlight: June 1, 2016

Researchers improve upon new gene editing technique

“Base Editor” corrects point mutations more reliably

National Institutes of Health-funded researchers unveiled a new technique that may help correct DNA mutations, the alterations in gene coding that can lead to various diseases. The new gene editing system improves upon recently heralded DNA editing methods, and it may be more efficient at correcting point mutations, the type of alteration that causes many genetic diseases. The group successfully used the novel approach to correct mutations related to Alzheimer’s disease and cancer in human and mouse cells. The new research is supported by the National Institute of Biomedical Imaging and Bioengineering (NIBIB), part of NIH.

Modified enzymes effect changes in bases of DNA in new gene editing technique.

“It’s a very exciting step forward,” said David Rampulla, Ph.D., program director for the Division of Discovery Science and Technology at NIBIB. “It makes a recent breakthrough even more powerful and specific to targeting harmful mutations.”

The most promising method for editing DNA has been the CRISPR/Cas9 system, a recent advance that snips DNA at specific target sequences, cutting out the segment that includes the mutation. Short segments with the correct sequence are added, with the hope that the cell will insert the new, correct one into the gap. However, sometimes the cell just patches together the two cut ends, making random insertions and deletions to force the strand back together. For correcting point mutations, where just one DNA base is altered, the current CRISPR/Cas9 system works less than 5 percent of the time.

“The majority of mutations thought to drive genetic disease in humans are point mutations,” said David Liu, Ph.D., Howard Hughes Medical Institute Investigator at Harvard and senior author of the paper. “But current standard genome editing methods aren’t particularly good at correcting point mutations.”

Instead, Liu and colleagues created a more direct editing method; they modified the CRISPR system so that, rather than removing a small section of DNA, it converted one chemical base into another. DNA is a double-stranded molecule that has four bases: cytosine (C) on one strand binds guanine (G) on the other, while thymine (T) and adenine (A) form the other binding pair. The new approach, termed “base-editing,” switched a C-G pair into a T-A pair.

The work was performed by researchers from the department of chemistry and chemical biology at Harvard University in Cambridge, Massachusetts. The results were reported online on April 20, 2016 in Nature.

Base Editing

The team started with a version of the Cas9 enzyme with its cutting abilities disabled; it would form a bond with the target sequence but leave both DNA strands intact. To this version of Cas9, they added another enzyme (APOBEC1) that converts C into uracil (U). U is naturally found only in RNA, but acts similarly to the T in DNA; so it will also form a bond with A. The hope was that the cell would see a mismatch (the new U paired with the original G) and fix it by converting the G to an A.

Although the method worked well in a test tube, the efficiency plummeted when the group tried it in human cells. Liu anticipated it wouldn’t be easy. He told Alexis Komor, his postdoctoral researcher and first author of the paper, “Even after we develop a base editor that works well in a test tube, when we try to move it into a cell, we’re going to have a real fight on our hands.”

Because of both the presence of a U and the incorrect bond with a G, the cell’s impulse is to immediately change the U back to a C. To overcome both of these issues, the team made two more modifications to the enzyme complex. These additional modifications increased the efficiency in human cells to about 30 percent (up from less than 1 percent with the original Cas9), with minimal random insertions or deletions.

Lastly, the team attempted to use base editing to correct two disease-causing point mutations, one in a gene that increases Alzheimer’s disease risk (APOE4) and another that is associated with different types of cancer (p53). In experiments with mouse cells, the new method corrected the APOE4 mutation 58-75 percent of the time. In experiments with human cells, it corrected the p53 mutation about 3-8 percent of the time. Depending on the disease, Liu says 100 percent efficiency may not be necessary; for some disorders, only a small percentage of correction could potentially ameliorate the disease. Importantly, both conversions were much more efficient than the original CRISPR/Cas9 system, which corrected APOE4 about 0.2 percent of the time and was never successful for p53.

Now the group is trying to find an enzyme—or a way of making one—that will convert other bases and they’re hoping to apply the same principle to other mutations. Eventually, after many more optimizations and large-scale safety studies, the method may find its way to the clinic.

“We hope that it will help expand the efficiency and cleanliness of genome editing, for the largest class of human mutations that drive disease, namely the point mutations,” said Liu. “That said, there’s still much additional work that has to be accomplished before base editing will be able to address any human diseases.”